Chromatin Immunoprecipitation Analysis in Filamentous Fungi

Detail:

Series: Methods in Molecular Biology | Volume: 944 | Pub. Date: Nov-14-2012 | Page Range: 221-236 | DOI: 10.1007/978-1-62703-122-6_16

Year prepared: 2012

Chromatin immunoprecipitation (ChIP) is used to map the interaction between proteins and DNA at a specific genomic locus in the living cell. The protein–DNA complexes are stabilized already in vivo by reversible crosslinking and the DNA is sheared by sonication or enzymatic digestion into fragments suitable for the subsequent immunoprecipitation step. Antibodies recognizing chromatin-linked proteins, transcription factors, artificial tags, or specific protein modifications are then used to pull down DNA–protein complexes containing the target. After reversal of crosslinks and DNA purification locus-specific quantitative PCR is used to determine the amount of DNA that was associated with the target at a given time point and experimental condition. DNA quantification can be carried out for several genomic regions by multiple qPCRs or at a genome-wide scale by massive parallel sequencing (ChIP-Seq).

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